Burkholderia mallei lipopolysaccharide (LPS) has been previously shown to cross-react with polyclonal antibodies raised against B. pseudomallei LPS; however, we observed that B. mallei LPS does not react with a monoclonal antibody (Pp-PS-W) specific for B. pseudomallei O polysaccharide (O-PS). In this study, we identified the O-PS biosynthetic gene cluster from B. mallei ATCC 23344 and subsequently characterized the molecular structure of the O-PS produced by this organism.Burkholderia mallei is a gram-negative bacterium responsible for a disease known as glanders in solipeds and occasionally in humans (3,8,13). The factors involved in the pathogenesis of B. mallei infection remain relatively poorly defined at the molecular level. A previous study that identified a polysaccharide gene cluster in B. mallei showed that B. mallei lipopolysaccharide (LPS) cross-reacts with polyclonal antibodies raised against the LPS of Burkholderia pseudomallei, a closely related organism responsible for a disease known as melioidosis (6). In the present study, we investigated the LPS profiles of B. mallei strains, identified the gene cluster responsible for O polysaccharide (O-PS) biosynthesis in B. mallei ATCC 23344, and determined the physical structure of the B. mallei ATCC 23344 O-PS. Additionally, we showed that the O-PS moiety of B. mallei LPS is required for resistance to the bactericidal action of serum. Finally, we identified the presence of insertion sequences in two strains of B. mallei that disrupt the expression of O-PS.Analysis of LPS profiles of B. mallei strains. The strains and plasmids used in this study are shown in Table 1. The first goal of this study was to assess the LPS profiles of B. mallei strains. Initially, we performed Western blot analysis of B. mallei ATCC 23344 whole-cell lysates with polyclonal rabbit sera raised against a B. pseudomallei bovine serum albumin (BSA)-O-PS conjugate as well as with a B. pseudomallei O-PS-specific MAb (Pp-PS-W) according to a previously described protocol (1, 2). As shown in Fig. 1A, B. mallei ATCC 23344 reacted with the anti-LPS polyclonal sera, resulting in a typical LPS banding pattern; however, the B. pseudomallei O-PS-specific MAb (Pp-PS-W) did not react. This indicated that differences exist between B. mallei and B. pseudomallei O-PS. We further assessed the LPS profiles of 10 different B. mallei strains (Fig. 1B). By using Western blot analysis, we showed that 8 of the 10 strains assessed bound the anti-LPS polyclonal sera and displayed typical LPS banding patterns. In contrast, however, two strains, NCTC 120 and ATCC 15310, did not bind the anti-LPS polyclonal sera, as indicated by the absence of bands (Fig. 1B). In order to confirm that the O-PS moiety was absent rather than a different type of O-PS, silver stain analysis was employed. Figure 1C shows the silver stain results confirming that both of these strains lacked O-PS moieties.Identification and characterization of B. mallei ATCC 23344 O-PS biosynthetic gene cluster. In order to investigate the genes re...